Bile acids conjugates with metal ion chelates and the use thereof

ABSTRACT

Compounds able to chelate paramagnetic metal ions and the use thereof as contrast agents in the technique known as “Magnetic Resonance Imaging” (M.R.I.).

This application is the U.S. national phase of international applicationPCT/EP01/01971 filed 21 Feb. 2001 which designated the U.S.

The present invention relates to novel compounds able to chelateparamagnetic metal ions and to the use thereof as contrast agents in thetechnique known as “Magnetic Resonance Imaging” (M.R.I.). In particular,the present invention relates to compounds resulting from theconjugation of a carrier consisting of a bile acid residue withmolecules endowed with a chelating capacity, as well as their complexchelates with bi- and trivalent paramagnetic metal ions, the saltsthereof and the use thereof as contrast agents for M.R.I.

Patent literature reports a number of patents and patent applicationsconcerning chelates of straight and cyclic polyaminopolycarboxylicligands with paramagnetic metals as contrast agents for M.R.I. Some ofthese compounds are already in clinical use (Gd-DTPA, N-methylglucaminesalt of the gadolinium complex with diethylenetriaminopentaacetic acid,MAGNEVIST®, Schering; Gd-DOTA, N-methylglucamine salt of thegadolinium/1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidcomplex, DOTAREM®, Guerbet; Gd-HPDO3A, gadolinium complex with10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, PROHANCE®, BRACCO). These contrast agents are designed for awholly general use, as they are mainly distributed in the extracellularspaces after administration.

On the contrary, a contrast agent capable of providing diagnosticallyuseful information even in case of small lesions such as liver tissuemetastasis, should selectively accumulate in the intracellular spaces ofthe parenchyma of said organ. Intracellular hepatobiliary agents arecapable of entering hepatocytes through the sinusoid membrane and aremainly excreted through the bile. The lipophilic nature of theirchelating units is considered responsible for the preferential uptake byhepatocytes (Lauffer, R. B.; Chem. Rev. 87, 901-927, 1987; Lauffer, R.B. et al.; Magn. Reson. Med. 4, 582-590, 1987).

Cholic acid is one of the bile acids resulting from cholesterolcatabolism; it is excreted by hepatocytes into bile (Elliot, W. H.;Sterols and bile acids; H. Danielsson and J. Sjövall, Eds.; pp 231-278,Elsevier, N.Y., 1985).

M.R.I. contrast agents comprising a residue of a chelating agentconjugated by a spacing chain to a bile acid residue are disclosed inWO95/01958. Said agents are stated to be particularly useful in theimaging of liver and bile ducts.

It has also been found that the bile acid residue makes the chelantingagents including it capable of interacting with plasma proteins thusforming non-covalent bonds with them. Therefore the contrast agentscomprising a bile acid residue are also particularly useful in theN.M.R. imaging of the vasal system, as disclosed in Italian patentapplication MI98A002802, which is herein incorporated by reference inits entirety.

EP 279,307 (Abbott) discloses other polyaminopolycarboxylic chelants,capable of complexing metal ions, conjugated with different substrates,inter alia bile acids, as contrast agents. The only compound disclosedin this patent application is a ¹¹¹In complex of a conjugate in which afunctionalized derivative of EDTA is linked, through an amido bond, tothe carboxylic function of a cholic acid. The possibility of chelatingparamagnetic metal ions for use in M.R.I. is not referred to in any way.

Betebenner David A. et al. in Bioconjugate Chem. 2; 117-123, 1991disclose the preparation and characterization of an EDTA derivativeconjugated with a cholic acid and of the corresponding chelated complexwith ¹¹¹In. The conjugation takes place through the terminal amino groupof a functional derivative of EDTA with a cholic acid ester, by means ofa reaction already used for linking chelating units to proteins(Westerberg, D. A. et al., J. Med. Chem. 32, 236-243, 1989; Brechbiel,M. W. Et al., Inorg. Chem. 25, 2772-2781, 1986). Biodistribution andscintigraphic imaging studies carried out with this compound show that,after uptake by the liver, it rapidly passes into the intestine.

All known agents comprising the residue of a bile acid are characterizedby the presence of a single chelating unit and, therefore, of a singlemetal ion coordinated per each molecule of the agent itself.

The present invention relates to compounds capable of chelating at leasttwo metal ions. Said compounds are obtained by conjugation of a carrierconsisting of a bile acid residue with two chelating molecules, saidconjugation taking place through a central reactive polyfunctionalsubstrate or central synton. In particular, the bonds between thecentral syntons of the invention and the chelating units are obtained bymeans of functional residues suitably selected so that said chelatingunits can keep unaltered their capability to coordinate the metal ionand the stability of the resulting chelates does not decrease.

More particularly, the present invention relates to compounds of generalformula (I)

wherein:

A is a polyfunctional reactive substrate containing at least threefunctional groups deriving from any polyvalent organic residue which canbe aliphatic with open chain, optionally branched, or alicyclic, orheterocyclic containing N, O and/or S, or aromatic or heteroaromatic;

Z is a bile acid residue;

Y₁ and Y₂, which can be the same or different, are the residue of achelating agent of the bi-trivalent metal ions having atomic numbersranging between 20 and 31, 39, 42, 43, 44, 49, and between 57 and 83;

L₁, L₂ and L₃, which can be the same or different, are a single bondbetween the functional groups Y1and A, Y2 and A and/or Z and A, or aspacer comprising at most 20 carbon atoms.

Bile acid herein means all bile acids obtained by bioconversion orsynthetic modification of cholesterol, particularly cholic, deoxycholic,chenodeoxycholic, ursodeoxycholic, lithocholic acids and the derivativesthereof, including those with taurine and glycine. Said residues arelinked to the L₃ group through covalent bonds involving the hydroxygroup, optionally converted to an amino group, at the 3-position or thecarboxy group at the 24-position of the cholane skeleton.

Preferred compounds are those in which L₁ and L₂, which can be the sameor different, are a spacer chain of formula (II)

wherein:

Q is a C₁-C₈ alkyl chain optionally substituted with 1 to 3 OH groups;

s is an integer 0 to 5;

R₁ is an H atom, or a C₁-C₅ alkyl group;

X is

L₃ is a group of formula (III)

in which: X and s have the above defined meanings (with the proviso thatwhen s is different from 0 then CO and X are present).

Particularly preferred are the compounds in which L₁ and L₂ are selectedfrom those of formula (IV), (V), (VI)

L₃ is a single bond between the Z and A functional groups or a group

in which:

n is an integer 1 to 8, and

R₁ has the above defined meanings.

The invention also relates to the complex chelates of said compounds offormula (I) with the bi- and trivalent ions of the metal elements havingatomic numbers ranging between 20 and 31, 39, 42, 43, 44, 49, andbetween 57 and 83, as well as the salts thereof with physiologicallycompatible organic bases selected from primary, secondary, tertiaryamines or basic amino acids, or with inorganic bases the cations ofwhich are sodium, potassium, magnesium, calcium or mixtures thereof, orwith anions of physiologically acceptable organic acids selected, forexample, from acetate, succinate, citrate, fumarate, maleate, oxalate,or with anions of inorganic acids such as ions of halo acids, i.e.chlorides, bromides, iodides.

The invention also relates to the preparation of the compounds ofgeneral formula (I) as well as the complex salts thereof and the usethereof for the preparation of pharmaceutical formulations for thediagnostic use, in particular for M.R.I. contrast formulations.

Furthermore, the compounds of the invention can optionally be chemicallyconjugated with suitable macromolecules or englobated in suitablecarriers.

Preferred compounds are those in which Z is a residue deriving from thefollowing bile acids or derivatives thereof with taurine and glycine:

A is a polyfunctional reactive substrate having preferably amino and/orcarboxy groups. Preferred examples of substrates A are reported in Table1.

TABLE 1

The compounds 1-17 can be used as such or as amino- or carboxy-protectedor activated derivatives.

Y₁ and Y₂ are preferably the residues of two polyaminopolycarboxylicligands, either in the form of acids or of derivatives thereof. Moreparticularly, Y₁ and Y₂ are preferably residues ofdiethylenetriaminopentaacetic (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic (DOTA),10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic(HPDO3A),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic(BOPTA) acids, or ofN-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethyl]glycine(EOB-DTPA) andN,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]-ethyl]glycine(DTPA-BMA).

Particularly preferred are the compounds in which Z is a cholic acidresidue, A is a polyfunctional substrate selected from those of formulae1, 2, 3, 4 and 17 and Y₁ and Y₂, which can be the same or different, areselected from residues shown in the following Table 2:

TABLE 2

residue a

residue b

residue c

residue d

residue e

residue f

Particularly preferred are the following compounds:

[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)-amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid;

[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-2-carboxyethyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid;

(3α,5β,12α)-3-[[[trans-3,4-bis[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid;

[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[[2-[bis(carboxymethyl)-amino]ethyl](carboxymethyl)amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid;

[3α[1(S),2(S)],5β,7α12α]-3-[[[[cis-1,2-bis[[[5-[bis[2-[bis(carboxymethyl)-amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-4-cyclopentyl]amino]-carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid;

[3α[1(S)],5β,7α,12α]-3-[[[[cis-1-[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-5-carboxypentyl]amino]carbonyl]-2-[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-4-cyclopentyl]amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid;

[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxy-cholan-24-oicacid;

as well as the chelates and the physiologically compatible saltsthereof.

Suitable ions for forming complex salts with the chelating agents ofgeneral formula (I) are bivalent or trivalent ions of the elementshaving atomic numbers ranging between 20 and 31, 39, 42, 43, 44, 49,between 57 and 83; particularly preferred are Fe⁽²⁺⁾, Fe⁽³⁺⁾, Cu⁽²⁺⁾,Cr⁽³⁺⁾, Gd⁽³⁺⁾, Eu⁽³⁺⁾, Dy⁽³⁺⁾, La⁽³⁺⁾, Yb⁽³⁺⁾, or Mn⁽²⁺⁾, or alsoradioisotopes such as ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb,¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi.

The compounds of the present invention have shown good tolerability aswell as water solubility.

The improvement of the M.R.I. imaging that the radiologist can observe,namely an increase in the contrast between healthy and affected tissues,is certainly an aid for diagnosis. Said improvement is generallyobtained by administering the patient with suitable exogenous compounds,such as the suitably complexed paramagnetic metal ions, capable ofsignificantly changing the relaxivity of the water protons of the tissuethey are in contact with, when said protons are subjected to an outermagnetic field.

The change induced by the presence of two paramagnetic metal ions permolecule of the administered agent, characteristic of the compounds ofthe invention, is significantly enhanced thereby obtaining a contrastbetween healthy and affected tissue which is already diagnosticallyuseful at lower dosages of contrast agents.

An advantage of the complex chelates of the invention is therefore thatthey can be used in low-dosage diagnostic formulations capable ofproviding good-quality images while remarkably decreasing the toxicityand the discomfort unavoidably involved by the administration of anexogenous substance, such as a contrast agent.

The compounds of the invention can therefore be advantageouslyadministered at doses ranging from 0.005 to 1.0 mmol/kg, and preferablyfrom 0.01 to 0.1 mmol/kg.

Thanks to the presence in the molecule of a carrier such as the bileacid residue, the agents of the invention can interact with plasmaproteins and form non-covalent bonds therewith. This property thusprovides high relaxivity in the serum and such long permanency in bloodas to make them particularly suitable for M.R.I. imaging of the vascularsystem.

It has surprisingly found that, in addition to increasing the number ofthe paramagnetic metal ions present, which involves advantages in termsof relaxivity and signal intensity, the introduction of a secondchelating unit in the compounds of the invention induces a remarkablechange in the excretion mechanism thereof. The compounds of theinvention, in fact, are surprisingly almost completely excreted thoroughthe renal route, by glomerular filtration. The experimental results ofthe pharmacokinetic screening in rats indicate that during a period of 0to 480 min, 36.2% of the administered compound was eliminated with urineand only 0.175% of the injected dose with bile. A detailed descriptionof the experimental conditions as well as of the obtained results isincluded, as a non-limiting example, in the experimental section.

The compounds of the invention, therefore, while maintaining unaffectedthe properties deriving from the presence of the bile acid residue, donot enter liver intracellular spaces and are excreted through the bileonly to a very limited extent, contrary to the compounds of the priorart containing one chelant residue only.

This involves, on the one hand, an unexpected improvement in terms oftoxicity and, on the other hand, total absence of metabolization byhepatocytes.

From still another point of view, the agents of the invention haveremarkably increased half-life in blood, as they are captured andenglobated by the hepatocytes only to a very low extent. This furtherunexpected property, as well as the increased relaxivity in serum,contribute to make the agents of the present invention particularlysuitable for the imaging of the vascular system in general, andparticularly of coronaries, even at the lows dosages used.

The compounds of general formula (I) can be prepared with syntheticmethods usually employed in industrial technique.

In particular, the preparation of compounds in which the two chelatingunits are the same is performed according to a synthetic process similarto that illustrated in the following Scheme, representing the synthesisof the compound of Example 1, as detailed in the experimental section.

Briefly, the synthetic process of the scheme comprises the followingsteps:

1) synthesis of the central polyfunctional residue A in a suitable form,in which the functional groups can independently be suitably protectedor activated;

2) synthesis of the suitably functionalized chelant residue B, i.e. of achelating agent capable of stably coordinating the metal ions as well asof covalently binding to the central polyfunctional residue through asuitable functional group;

3) linking between central polyfunctional residue and chelant residuesand isolation of intermediate C;

4) synthesis of a suitably functionalized bile acid D capable of stablybinding to the central polyfunctional residue through a suitablefunctional group;

5) optional cleavage and/or activation of the third functional residueA, linking between the resulting compound and D and isolation of E;

6) cleavage of any protective groups and isolation of polyacid F;

7) complexation of the metal ions and isolation of the chelated complexG.

When the two chelating units of the compounds are different, thecontrast agents of the invention can be prepared by using a similarsynthetic process in which intermediate C is prepared by reacting thesuitable polyfunctional residue A with a first chelant residue B1,subsequent activation of a second functional group A and linking betweenit and the second chelant residue B2.

Briefly, said process comprises the following synthetic steps:

1) synthesis of the central polyfunctional residue A in a suitable form,in which the functional groups can independently be suitably protectedor activated;

2) synthesis of the two suitably functionalized chelant residues B1 andB2, i.e. of two chelant units capable of stably coordinating the metalions as well as of covalently binding to the central polyfunctionalresidue through a suitable functional group;

3) linking between polyfunctional residue and a first chelant residueB1; optional cleavage and/or activation of a second functional residueA, linking between it and a chelating unit B2 and isolation ofintermediate C;

4) synthesis of a suitably functionalized bile acid D, capable of stablybinding to the central polyfunctional residue through a suitablefunctional group;

5) optional cleavage and/or activation of the third functional residueA, linking between the resulting compound and D and isolation of E;

6) cleavage of any protective groups and isolation of polyacid F;

7) complexation of the metal ions and isolation of the chelated complexG.

Said compounds, or the suitable precursors and/or functional derivativesthereof, can be easily obtained according to the known synthetictechniques.

By way of example, compound 2 is commercially available, derivatives ofcompound 7 in which the amino groups are suitably protected can beprepared as described in J. Am. Chem. Soc. 1996, 118, 2567 or in Polymer1982, 23, 771; compound 5 as described in Tetrahedron Lett. 1993, 34,4989; compound 4 dimethyl ester as described in J. Org. Chem. 1989, 54,5115; compound 1 dimethyl ester with the amino residue suitablyprotected as benzyl derivative, as described in Bull. Soc. Chim. Fr.1988, 579; compound 3 derivative in which the amino functions areappropriately protected, as described in Chem. Commun. 1998, 1629;compounds 6 and 8 dimethyl esters in which the amino functions aresuitably protected, as described in J. Chem. Soc. Perkin trans. I 1991,409 and Tetrahedron Lett. 1994, 35, 4515 respectively; compound 13dimethyl ester as described in J. Med. Chem. 1990, 33, 1561; anhydride17 as described in Chem. Pharm. Bull. 1984, 32, 805.

The residues of Table 2 can be prepared using known techniques. Theresidues a, b and c can, for example, be prepared analogously to whatdescribed in Bioconjugate Chem. 1999, 10, 137; the preparation of theresidue d is described in WO 9601655, that of the residue e in WO9528967, that of f is described in the experimental section, Example 4.

Preferred bile acid functional derivatives for the preparation of thecompounds of the invention are, for example, the 3β-amino derivativesthereof, obtained starting from corresponding 3α-hydroxyls following thesynthetic process described in Synth. Commun. 1998, 28, 109.

Also preferred are chloroorthoformates obtained from the corresponding3α-hydroxyls according to the synthetic process described in J. Am.Chem. Soc. 1997, 117, 640.

Both cited derivatives can be used in the linking reactions directly, orthey can be further modified by reacting the respective functionalgroups with suitable bifunctional spacers.

A list of particularly preferred bile acid functional derivatives isreported hereinbelow. For many compounds, the list also reports abibliographic reference concerning the preparation thereof.

Bile Acid Functional Derivatives

The linking between polyfunctional central nucleus and chelant residuecan take place, for instance, between a suitably functionalized aminogroup present on the chelant and a carboxylic residue present on thepolyfunctional central residue or, vice versa, between a carboxylicresidue of the suitable ligand functional derivative and an aminoresidue of the central polyfunctional residue to form an amide in bothcases.

The linking between the central polyfunctional residue and the bile acidresidue can take place by formation of an amido bond, for examplebetween the acid group at the 24-position of the bile residue and anamino group present in the central polyfunctional synton, according to aprocedure widely described and used in the preparation of, for example,peptides (Tetrahedron, 32, 2211, 1976).

Alternatively, the amido bond can be formed between the amino group of abile acid 3β-amino derivative and a carboxylic group of the centralpolyfunctional residue, or between the chlorocarbonyl residue of thebile acid chloroformate and an amino group of the central polyfunctionalresidue.

Said linking may optionally be preceded by cleavage and/or activation ofthe functional group of the central unit involved in the reaction.

Amino functional groups are usually protected by transformation into thecorresponding benzyl, carbobenzyloxy (Cbz) or tert-butyloxycarbonyl(BOC) derivatives. In the first two cases, for example, the cleavagereaction can take place by hydrogenation of the benzyl or Cbzderivatives, in the presence of a suitable metal catalyst, for examplePd/C. In the third case, cleavage can be obtained in acidic conditions,for example with CF3COOH.

The carboxylic residues are usually protected by transformation intosuitable esters. The subsequent deprotection of carboxylic functions ofthe two chelant units can be obtained by hydrolysis of the protectiveester groups, for example with LiOH, CF3COOH or iodotrimethylsilane in asuitable organic solvent, such as dioxane, CH₂Cl₂ or CH₃CN or analcohol, such as i.PrOH.

The compounds of the invention have a wide range of applications, sincethey can be administered through the intravasal (for instanceintravenous, intraarterial, intracoronaric, intraventricular routes, andso on), intrathecal, intraperitoneal, intralymphatic and intracavitalroutes. The compounds are also suitable for the oral or enteraladministration, and therefore, for the imaging of the gastrointestinaltract.

For the parenteral administration, the compounds of the invention arepreferably formulated as sterile solution or aqueous suspension, with pHpreferably ranging from 6.0 to 8.5. Said aqueous solutions orsuspensions can be administered in concentrations ranging from 0.002 to1.0 molar. The resulting formulations can be lyophilized and supplied assuch, to be reconstituted prior to use.

For the gastrointestinal use or for injection to body cavities, theseagents can be formulated as a solution or suspension containing suitableadditives in order to, for example, control viscosity.

For the oral administration they can be formulated according topreparation methods routinely used in the pharmaceutical technique or ascoated formulations to gain extra protection from the acid pH ofstomach, inhibiting the release of the chelated metal ion, which usuallyoccurs at typical pH values of gastric juices.

Other excipients, such as sweeteners and/or flavoring agents, can bealso added according to known techniques of pharmaceutical formulation.

The solutions or suspensions of the compounds of this invention can alsobe formulated as aerosol to be used in aerosol-bronchography andinstillation.

As far as diagnostic imaging is concerned, the chelates of thisinvention can also be used as radiopharmaceuticals in nuclear medicineboth in the diagnostic and therapeutic field. However, in this case themetal ion which is chelated is a radioisotope, such as ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga,¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc,¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi.

Preferred cations of inorganic bases which can be suitably used tosalify complex chelates of this invention particularly comprise ions ofalkali or alkaline-earth metals such as potassium, sodium, calcium,magnesium and the mixtures thereof.

Preferred cations of organic bases suitable for the above mentioned aim,comprise, among others, those of primary, secondary and tertiary aminessuch as ethanolamine, diethanolamine, morpholine, glucamine,N-methylglucamine, N,N-dimethylglucamine.

Preferred anions of inorganic acids which can be suitably used for thesalification of complex chelates of this invention particularly compriseanions of the hydrohalo acids such as chlorides, bromides, iodides orother anions such as sulfate.

Preferred anions of organic acids suitable for the above mentioned aimcomprise those of acids routinely used in pharmaceutical technique forthe salification of basic substances, such as acetate, succinate,citrate, maleate and fumarate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine, ornithine or of aspartic andglutamic acids.

The compounds of the invention can be encapsulated or in liposomes orform the constituents of their chemical structure and used as uni- ormultilamellar vesicles.

The compounds of the invention can also be conjugated withmacromolecules or englobated into or combined with suitable carriers.

In the following, a non-limiting list of preferred compounds of theinvention is reported.

COMPOUND 1 (EXAMPLE 1)

COMPOUND 2 (EXAMPLE 2)

COMPOUND 3 (EXAMPLE 3)

COMPOUND 4 (EXAMPLE 4)

COMPOUND 5 (EXAMPLE 5)

COMPOUND 6 (EXAMPLE 6)

COMPOUND 7 (EXAMPLE 7)

EXPERIMENTAL SECTION EXAMPLE 1

Gadolinium complex of[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid salified with sodium (1:5)

a) trans-1-(phenylmethyl3,4-pyrrolidinedicarboxylic acid.

Trans-1-(phenylmethyl)-3,4-pyrrolidinedicarboxylic acid dimethyl ester(13.4 g; 48.3 mmol) (prepared as described in Bull. Soc. Chim. Fr. 1988,579) is dissolved in a mixture of EtOH (220 mL) and H₂O (35 mL) andsaponified with 1N NaOH (96.4 mL). The solution is then concentrated andthe residue taken up into 100 mL of H₂O. The solution is acidified to pH1.8 with 6N HCl to precipitate the acid which is isolated as a whitesolid (10 g; 40.11 mmol).

Yield: 83%

M.p.: 140-145° C.

HPLC assay: 99.5% (in % area)

Elemental analysis:

% calc.: C 62.24; H 6.07; N 5.62.

% found: C 63.77; H 6.15; N 5.63.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b)N²,N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-N⁶-[(phenylmethoxy)carbonyl]-L-lysinemethyl ester.

N⁶-[(phenylmethoxy)carbonyl]-L-lysine methyl ester hydrochloride (10.6g; 32.2 mmol) (commercial product) andN-(2-bromoethyl)-N-[2-(1,1-dimethylethoxy)-2-oxoethyl]glycine1,1-dimethylethyl ester (prepared according to Rapoport, J. Org. Chem.1993, 58, 1151) (27.2 g; 77.1 mmol) are dissolved in CH₃CN (160 mL). Themixture is then added with buffer phosphate 2M pH 8 (160 mL) and stirredfor 2 hours. The phases are then separated, the aqueous layer isreplaced with fresh buffer (160 mL) and the mixture is stirred for afurther 70 hours. The organic phase is then separated and evaporated,and the residue is taken up into CH₂Cl₂ (150 mL). The resultingmethylene solution is washed with H₂O and evaporated to a residue toobtain an oil which is purified by silica gel chromatography. Thehomogeneous fractions are evaporated to obtain the title compound as ayellow oil (18.1 g; 21.7 mmol).

Yield: 67%

HPLC assay: 97.8 (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt=1:1.

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.58

Elemental analysis:

% calc.: C 61.70; H 8.67; N 6.69;

% found: C 61.85; H 8.74; N 6.34.

Specific rotatory power: [α]_(D) ²⁰=−27.05; (c 5.1, CHCl₃).

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c)N²,N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysinemethyl ester

A solution of the intermediate from step b) (40 g; 47.5 mmol) in MeOH(700 mL) is added with carbon on Palladium (4 g; 5% Pd). The suspensionis stirred at room temperature under hydrogen atmosphere for 4 hours,then filtered through Millipore® HA 0.45 μm filter and the catalyst iswashed with MeOH. The solution is evaporated to obtain the desiredproduct as yellow oil (32.5 g; 46.2 mmol).

Yield: 97%

HPLC assay: 99 (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt=1:1.

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.13

Elemental analysis:

% calc.: C 59.80; H 9.46; N 7.97; H₂O

% found: C 57.89; H 9.44; N 7.39; H₂O 0.98.

Specific rotatory power: [α]_(D) ²⁰=−28.68; (c 5.0, CHCl₃).

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

d) N⁶,N⁶′-[[trans-1-(phenylmethyl)-3,4-pyrrolidinediyl]biscarbonyl]bis[N²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine]dimethyl ester.

A solution of the compound from step a) (4.0 g; 16.0 mmol) in CH₃CN (110mL), cooled to 0° C. and kept under nitrogen atmosphere, is added withtriethylamine (4.9 mL; 35.3 mmol) and then pivaloyl chloride (4.4 mL;35.3 mmol). After 20 min the resulting mixture is added dropwise with asolution of the compound from step c) (24.8 g; 35.3 mmol) in CH₃CN (90mL). The mixture is then warmed at room temperature and kept under theseconditions for 1 h, then evaporated. The residue is taken up into AcOEt(300 mL), washed with H₂O and concentrated. The resulting crude oil ispurified by silica gel chromatography to obtain the title product asyellow oil (17.5 g; 10.9 mmol).

Yield: 68%

HPLC assay: 98.4% (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt=1:1.

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.24

Elemental analysis:

% calc.: C 61.57; H 8.90; N 7.79;

% found: C 61.58; H 8.99; N 7.48.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

e)N⁶,N⁶′-[[trans-3,4-pyrrolidinediyl]biscarbonyl]bis[N²N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine]dimethyl ester.

A solution of compound from step d) (21.6 g; 13.4 mmol) in EtOH (250 mL)is added with carbon on palladium (2.2 g; 5% Pd) and the suspension iskept 4 hours under hydrogen atmosphere, with strong stirring. Themixture is then filtered through a Millipore® FH 0.5 μm filter andevaporated to a residue to obtain the reduction product as yellow oil(18.2 g; 11.9 mmol).

Yield: 90%

HPLC assay 99% (in % area)

Elemental analysis:

% calc.: C 59.70; H 9.03; N 8.24;

% found: C 59.12; H 9.38; N 7.72.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

f) (3α,5β,12α)-3-[(chlorocarbonyl)oxy]-12-hydroxycholan-24-oic acidmethyl ester.

A solution of 20% phosgene in toluene (100 mL) is dropped into asolution of (3α,5β,12α)-3,12-dihydroxycholan-24-oic acid methyl ester(14.7 g; 36 mmol) (commercial product) in dry CH₂Cl₂ at 0° C. and undernitrogen atmosphere. The solution is stirred for 3 h then evaporated toa residue to obtain the desired product as a white solid (15.2 g; 32.4mmol).

Yield: 90%.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

g)[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis[2-(1,1-dimethyl-ethoxy)-2-oxoethyl]amino]ethyl]amino]-6-methoxy-6-oxohexyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid methyl ester.

In a solution of the compound from step f) (5.5 g; 11.7 mmol) andN,N-diisopropylethylamine (4.5 mL) in CH₂Cl₂ (150 mL) cooled to 0° C.,is dropped, under nitrogen, a solution of the compound from step e)(17.8 g; 11.7 mmol) in CH₂Cl₂ (50 mL). The resulting mixture is stirredat room temperature for 3 hours, then washed with H₂O and concentrated.The residue is purified by flash chromatography to obtain the titlecompound as yellow oil (17.0 g; 8.7 mmol).

Yield: 74%

HPLC assay: 94.4% (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt=1:1

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.29

Elemental analysis:

% calc.: C 62.46; H 9.10; N 6.43;

% found: C 62.15; H 9.16; N 6.32.

¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

h)[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)-amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid.

In a solution of the polyester from step g) (14.0 g; 7.1 mmol) in1,4-dioxane (190 mL) is dropped, at room temperature, a 2M LiOH aqueoussolution (190 mL). After 26 hours the solution is concentrated and addedwith 2M aqueous HCl to final pH 1.8 (175 mL) to precipitate the acidwhich is filtered, washed with water and dried to obtain the titleproduct as a white solid (8.7 g; 5.9 mmol).

Yield: 83%

M.p.: 210-215° C.

HPLC assay: 98.9% (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: CHCl₃/MeOH/aq. NH₄OH=5:4:2

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.38

Elemental analysis:

% calc.: C 54.74; H 7.33; N 8.57; H₂O

% found: C 51.72; H 7.51; N 8.27, H₂O 4.96.

Specific rotatory power: [α]_(D) ²⁰=+16.17; (c 1; NaOH 1M)

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

i) Gadolinium complex of[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]-carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid salified with sodium (1:5).

The ligand from step h) is suspended in water and dissolved by additionof NaOH (32 mL). The resulting solution is added with GdCl₃ (3.5 g; 9.6mmol) in H₂O (20 mL), keeping pH at 6.5 by addition of 1M NaOH (17.5mL). The mixture is kept one hour at room temperature then loaded intoan Amberlite XAD® 16.00 column eluting with CH₃CN/H₂O gradient. Thefractions containing the product are evaporated to obtain it as a whitesolid (8.3 g; 4.4 mmol).

Yield: 92%

M.p.: >300° C.

HPLC assay: 100% (in % area)

El. analysis:

% calc.: C 42.60; H 5.12; N 6.67; Gd 16.65; Na 6.09; H₂O

% found: C 37.68; H 5.90; N 5.90; Gd 14.67; Na 6.47; H₂O 9.90.

Specific rotatory power: [α]_(D) ²⁰=−12.32; (c 2; H₂O)

IR and MS spectra are consistent with the indicated structure.

EXAMPLE 2

Following the procedure described in example 1, starting from3-[[(phenylmethoxy)carbonyl]amino]-N-tert-butoxycarbonyl-L-alaninemethyl ester (Chem. Pharm. Bull. 1985, 33, 509), the gadolinium complexof[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[2-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-2-carboxyethyl]-amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxy-cholan-24-oicacid was prepared.

EXAMPLE 3

Following the procedure described in example 1, starting from1,4,7-tris-[2-(1,1-dimethylethoxy)-2-oxoethyl]-10-[[2-[2-aminoethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane(WO 95/28967, example 2), the gadolinium complex of(3α,5β,12α)-3-[[[trans-3,4-bis[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid was prepared.

EXAMPLE 4

Following the procedure described in example 1, using MnCl₂ for thecomplexation, the manganese complex of[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[[2-[bis(carboxymethyl)amino]ethyl](carboxymethyl)amino]-5-carboxypentil]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicwas prepared.

The chelant unit used in this case isN²-[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-N²-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-lysinemethyl ester, whose synthesis is described in the following:

a)N²-[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-N²-[2-(1,1-dimethylethoxy)-2-oxoethyl]-N⁶-[(phenylmethoxy)carbonyl]-L-lysinemethyl ester.

A solution ofN-(2-bromoethyl)-N-[2-(1,1-dimethylethoxy)2-oxoethyl]glycine1,1-dimethylethyl ester (21.1 g; 60 mmol) in CH₃CN (25 mL) is droppedinto a mixture of N⁶-[(phenylmethoxy)carbonyl]-L-lysine methyl esterhydrochloride (20 g; 60.4 mmol) (commercial product) andN,N-diisopropylethylamine (12.6 mL) in CH₃CN (250 mL). After 120 h atroom temperature, tert-butyl bromoacetate (18.8 g; 101.6 mmol) andN,N-diisopropylethylamine (17.7 mL) are added. The mixture is stirredfor a further 24 h, the solvent is then evaporated off and the residueis dissolved in Et₂O and filtered. The ether solution is washed with0.1M aq. HCl, dried and evaporated to a residue which is purified byflash chromatography. The homogeneous fractions are combined to obtainthe desired product as brown oil (13 g; 19 mmol).

Yield: 32%

HPLC purity: 91% (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt=7:3

Detection: 1% KMnO₄ in 1 M NaOH; Rf=0.4

Elemental analysis:

% calc.: C 61.83; H 8.45; N 6.18;

% found: C 61.70; H 8.52; N 5.84.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b)N²-[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-N²-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-lysinemethyl ester.

A solution of the compound from step a) (12.3 g; 18.1 mmol) in MeOH (120mL) is added with carbon on Palladium (1.3 g; 5% Pd) and the suspensionis kept 3 hours under hydrogen atmosphere, with strong stirring. Themixture is then filtered through a Millipore® FH 0.5 μm filter andevaporated to a residue to obtain the reduction product which is useddirectly.

EXAMPLE 5

Gadolinium complex of[3α[1(S),2(S)],5β,7α,12α]-3-[[[[cis-1,2-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5carboxypentyl]amino]-carbonyl]-4-cyclopentyl]-amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid salified with sodium (1:5)

a) N⁶,N⁶′-[(Cis-4-oxo-1,2-cyclopentanediyl)biscarbonyl]bis[N²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine]ditert-butyl ester.

A suspension of cis-tetrahydro-1H-cyclopenta[c]furan-1,3,5-trione (1.2g; 7.8 mmol) (obtained as described in Chem. Pharm. Bull. 1984, 32, 805)in THF (30 mL) kept at 20° C. is added dropwise with a solution ofN²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysinetert-butyl ester (5.8 g; 7.8 mmol) (Bioconjugate Chem. 1999, 10, 137) in20 mL THF. After 1h, the reaction mixture cooled to −5° C. is added withtriethylamine (2.17 mL; 15.57 mmol) and isobutylchloroformate (1 mL; 7.8mmol) (commercial product). The mixture is then warned to 20° C. and,after an hour,N²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysinetert-butyl ester (5.8 g; 7.8 mmol) in THF (25 mL) is dropped therein.The solution is kept at 20° C. for a further 18 h then concentrated. Theresidual oil is taken up into CH₂Cl₂ (75 mL) and H₂O (75 mL). Theorganic phase is separated, dried and purified by column flashchromatography to obtain the desired product (6 g; 3.7 mmol).

Yield: 47%

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: n-hexane/AcOEt/2-PrOH=7:2:1

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.29

Elemental analysis:

% calc.: C 61.31; H 9.17; N 6.89;

% found: C 61.02; H 9.04; N 6.83.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) N⁶, N⁶′-[(cis-4-idroxyimino-1,2-cyclopentanediyl)biscarbonyl]bis[N²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine]ditert-butyl ester.

A solution of the compound from step a) (14 g; 8.61 mmol) in H₂O (10 mL)and 2-propanol (150 mL), kept at 20° C., is added with hydroxylaminehydrochloride (2.69 g; 38.7 mmol) and sodium acetate (8.8 g; 64.6 mmol).The mixture is heated to 90° C. for 2 hours, concentrated and theresidue is taken up into CHCl₃ (100 mL) and H₂O (100 mL). Afterseparation of the phases, the aqueous solution is extracted with CHCl₃(200 mL). The combined organic phases are dried and concentrated toobtain the desired product as a white solid (11.8 g; 7.2 mmol).

Yield. 84%.

M.p.: 210-215° C.

HPLC assay: 96.8% (in % area)

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: CHCl₃/MeOH/Aq. NH₄OH=14:2:0.2

Detection: 1% KMnO₄ in IM NaOH; Rf=0.29

Elemental analysis:

% calc.: C 60.75; H 9.15; N 7.68; H₂O

% found: C 61.11; H 9.26; N 7.54; H₂00.29

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c)N⁶,N⁶′-[(cis-4-amino-1,2-cyclopentanediyl)biscarbonyl]bis[N²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine]ditert-butyl ester.

A solution of the compound from step b) (10 g; 6.1 mmol) in CH₃OH (60mL) is added with Ni-Raney (1.2 g) and the suspension is kept at 30° C.and 30 atm of hydrogen for 18 h. After cooling to 15° C. the solution isconcentrated and purified by flash chromatography to obtain the desiredproduct as oil (7.1 g; 4.4 mmol).

Yield: 72%.

HPLC assay: 97.1% (in % area)

K.F.: <0.1%

TLC: Carrier: silica gel plate 60F 254 Merck

Eluent: CHCl₃/MeOH/aq. NH₄OH=14:2:0.2

Detection: 1% KMnO₄ in 1M NaOH; Rf=0.52.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

d)[3α[1(S),2(S)],5β,7α,12α]-3-[[[[cis-1,2-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-4-cyclopentyl]amino]carbonyl]-oxy]-7,12-dihydroxycholan-24-oic.

Into a solution of(3α,5β,7α,12α)-3-[(chlorocarbonyl)oxy]-7,12-dihydroxycholan-24-oic acidmethyl ester (5 g; 10.3 mmol) (obtained as described in J. Am. Chem.Soc. 1997, 117, 640) and N,N-diisopropylethylamine (5 mL) in CH₂Cl₂ (150mL), cooled at 0° C., is dropped, under nitrogen, a solution of thecompound from step c) (16.8 g; 10.3 mmol) in CH₂Cl₂ (50 mL). Theresulting mixture is stirred at room temperature for 3 hours, thenwashed with H₂O and concentrated. The residue is purified by flashchromatography. The resulting product is dissolved in 1,4-dioxane (200mL) and an aqueous solution of 2M LiOH (200 mL) is dropped therein.After 24 h the solution is concentrated and acidified with 2M aq. HCl topH 1.8 to precipitate the acid, which is filtered, washed with water anddried to give the desired product as a white solid (6.8 g; 4.5 mmol).

Yield: 44%

HPLC assay: 98.4% (in % area)

Elemental analysis:

% calc.: C 54.43; H 7.32; N 8.40;

% found: C 54.33; H 7.25; N 8.35.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

e) gadolinium complex of[3α[1(S),2(S)],5β,7α,12α]-3-[[[[cis-1,2-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]-carbonyl]-4-cyclopentyl]amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid salified with sodium (1:5).

The ligand from step d) (6 g; 4 mmol) is suspended in water anddissolved with NaOH. The resulting solution is added with GdCl₃ (2.97 g;8 mmol) in H₂O (20 mL), keeping pH at 6.5 by addition of 1M NaOH. Themixture is kept 2 h at room temperature, then loaded on an AmberliteXAD® 16.00 column eluting with a CH₃CN/H₂O gradient. The fractionscontaining the product are evaporated to obtain the title compound as awhite solid (6.7 g; 3.5 mmol).

Yield: 88%

M.p.: >300° C.

HPLC assay: 100% (in % area)

Elemental analysis:

% calc.: C 42.56; H 5.15; N 6.57; Gd 16.39; Na 5.99;

% found: C 42.42; H 5.10; N 6.45; Gd 16.28; Na 5.87.

IR and MS spectra are consistent with the indicated structure.

EXAMPLE 6

Following the procedure described in example 5, starting fromcis-tetrahydro-1H-cyclopenta[c]furan-1,3,5-trione, 1,4,7-tris-[2-(1,1dimethylethoxy)-2-oxoethyl]-10-[[2-[2-aminoethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane(WO95/28967, example 2) andN²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysinetert-butyl ester (Bioconjugate Chem. 1999, 10, 137), the gadoliniumcomplex of[3α[1(S)],5β,7α,12α]-3-[[[[cis-1-[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-2-[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I-yl]acetyl]amino]ethyl]-amino]carbonyl]-4-cyclopentyl]amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid was prepared.

EXAMPLE 7

Gadolinium complex of[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]-carbonyl]amino]-7,12-dihydroxycholan-24-oicacid salified with sodium (1:5)

a) (3β,5β,7α,12α)-3-[[(3,5-diaminophenyl)carbonyl]amino]-7,12-dihydroxycholan-24-oic acid dihydrochloride methyl ester.

3,5-di(tert-butoxycarbonyl)aminobenzoic acid (3.16 g; 10 mmol) (preparedas described in Chem. Commun. 1998, 1629) is dissolved in DMF (150 mL)and the resulting solution is added, at 0° C., with triethylamine (3 mL)and diethylcyanophosphonate (1.8 g; 11 mmol) (commercial product). Intothe resulting mixture, a solution of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester(4.22 g; 10 mmol) (obtained as described in Synth. Commun. 1998, 28,109) is dropped, in 30 min. After 8 h at room temperature, the solutionis evaporated and the resulting residue is purified on silica gel byflash chromatography. The resulting product is treated withHCl-saturated MeOH (100 mL) to precipitate the title product as a whitesolid (2.7 g; 4.3 mmol).

Yield: 43%

HPLC assay: 97% (in % area)

Elemental analysis:

% calc.: C 61.14; H 8.18; N 6.68; Cl 11.28;

% found: C 60.98; H 8.11; N 6.59; Cl 11.22.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b)[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]-5-(1,1-dimethylethoxy)-1,5-dioxopentyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxycholan-24-oicacid.

A solution of the product from step a) (2.5 g; 4 mmol) in DMF,N,N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid tert-butyl ester (3 g; 4 mmol) (Bioconjugate Chem. 1999, 10, 137)and diethylcyanophosphonate (0.72 g; 4.4 mmol), kept at 0° C., isdropwise added with triethylamine (0.7 mL). After 1 h at 0° C. and 5 hat room temperature, the reaction mixture is evaporated and the residueis purified by flash silica gel chromatography, to obtain the desiredproduct as oil (5.79 g; 2.9 mmol).

Yield: 73%

HPLC assay: 98% (in % area)

Elemental analysis:

% calc.: C 64.18; H 9.10; N 4.94;

% found: C 64.03; H 8.99; N 4.95.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c)[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxy-cholan-24-oicacid.

Into a solution of the polyester from step b) (5.5 g; 2.8 mmol) in1,4-dioxane (100 mL) is dropped, at room temperature, a 2M LiOH aqueoussolution (100 mL). After 24 hours the solution is concentrated and addedwith 2M aq. HCl to final pH 1.8 to precipitate the acid which isfiltered, washed with water and dried to obtain the title product as awhite solid (3.6 g; 2.2 mmol).

Yield: 80%

HPLC assay: 99% (in % area)

Elemental analysis:

% calc.: C 61.92; H 5.93; N 5.95;

% found: C 61.85; H 5.88; N 5.90.

¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

d) gadolinium complex of[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxycholan-24-oicacid salified with sodium (1:5).

The ligand from step c) (3.5 g; 2.1 mmol) is suspended in H₂O anddissolved by addition of 1M NaOH. GdCl₃ (1.56 g; 4.2 mmol) in H₂O (20mL) is then dropped into the solution, keeping pH at 6.8 by addition of1M NaOH. The mixture is stirred for 4 h at room temperature then loadedon an Amberlite XAD® 16.00 column eluting with a CH₃CN/H₂O gradient. Thefractions containing the product are evaporated to obtain the titlecompound as a white solid (4.1 g; 2 mmol).

Yield: 95%

M.p.: >300° C.

HPLC assay: 100% (in % area)

Elemental analysis:

% calc.: C 49.39; H 4.19; N 4.74; Gd 15.21; Na 5.56;

% found: C 49.27; H 4.02; N 4.70; Gd 15.18; Na 5.48.

IR and MS spectra are consistent with the indicated structure.

EXAMPLE 8 Pharmacokinetics Screening in Rats

Materials and Methods

The experimentation was performed with the complex compound of example1.

The screening was performed in 3 anaesthetised CD(SD)BR male rats(Charles River Italia).

The animals were maintained according to EEC Council Directive 86/605 asincluded in Italian law DL116/92 (experimental typology KIN 3).

The contrast agent was injected in the vena saphena at the single doseof 0.1 mmol.kg⁻¹, injection rate of 6 mL-min⁻¹.

After the administration of the complex compound, the collection of thebiological fluids in the anaesthetised rats was performed by cannulationof urinary bladder and biliary duct. The urine was collected at 0 to 60,60 to 120, 120 to 240, 240 to 480 min after injection; the bile at −30to 0, 0 to 15, 15 to 30, 30 to 60, 60 to 120, 120 to 240, 240 to 480 minafter injection.

Animals were killed at the end of the studies by exanguination fromabdominal aorta.

Blood, liver and kidneys were collected after the sacrifice for theassay of gadolinium by inductively coupled plasma atomic emissionICP-AES.

Results

After intravenous administration to anaesthetised rats, gadolinium waseliminated both with urine and in trace amount with bile. In the 0 to480 min period, biliary and urinary excretion accounted for 0.175% and36% of ID, respectively. At 480 min after administration, the residualcontent in liver, kidney and plasma was 2.564%, 6.7%, and 14.3% of theinjected dose, respectively.

Biliary Urinary Collection time % of the injected dose % of the injecteddose 0 to 15 min 0.0102 ± 0.0010 0 to 30 min 0.0208 ± 0.0012 0 to 60 min0.0374 ± 0.0023 11.2 ± 4.6 0 to 120 min 0.0561 ± 0.0052 18.6 ± 5.0 0 to240 min 0.0950 ± 0.0073 27.9 ± 5.3 0 to 480 min 0.175 ± 0.020 36.2 ± 2.7

What is claimed is:
 1. A compound of formula (I):

wherein: A is a polyfurictional substrate selected from the following;

Z is a bile acid residue or a conjugate with taurine and glycine; Y₁ andY₂, which can be the same or different, are selected from the following:

residue a

residue b

residue c

residue d

residue e

residue f

L₁, L₂ and L₃, which can be the same or different, are a single bondbetween the functional groups of Y₁ and A, Y₂ and A and/or Z and A, or aspacer chain with at most 20 carbon atoms; as well as a complex chelatethereof with the ions of metal elements having atomic numbers rangingbetween 20 and 31, 39, 42, 43, 44, 49, and between 57 and 83, and a saltthereof with an organic base selected from primary, secondary, tertiaryamines or basic amino acids, or with an inorganic base the cations ofwhich are sodium, potassium, magnesium, calciuiin or mixtures thereof,or with anions of physiologically acceptable organic or inorganic acids;wherein linking between A and Y₁ and Y₂ is between a functionalizedamino group present on Y₁ and Y₂ and a carboxylic residue present on Aor between a carboxylic residue of Y₁ and Y₂ and an amino residue of Ato form an amide and wherein linking between A and the bile acid residueZ is by formation of an amido bond.
 2. A compound as claimed in claim 1wherein L₁ and L₂, which can be the same or different, are a spacingchain of formula (II)

wherein: Q is a C₁-C₈ alkyl chain optionally substituted with 1 to 3 OHgroups; s is an integer of 0 to 5; R₁ is an H atom, or a C₁-C₅ alkylgroup; X is

L₃ is a group of formula (III)

in which: X and s have the above defined meanings with the proviso thatwhen s is different from 0 then CO and X are present.
 3. A compound asclaimed in claim 1 in which L₁ and L₂, which can be the same ordifferent, are selected from those of formulae (IV), (V), (VI)

wherein: R₁ is an H atom, or a C₁-C₅ alkyl group and n is an integer of1 to 8 and L₃ is a single bond between Z and A or a group


4. A compound as claimed in claim 1 in which Z is a bile acid residueselected from one of the following:


5. A compound as claimed in claim 1, in which the bile acid residue Z isa cholic acid residue.
 6. A compound as claimed in claim 1 selected fromthe following:[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oicacid;[3α[3(S),4(S)],5β,12α]-3-[[[trans-3,4-bis[[[2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-2-carrboxyethyl]amino]carbonyl]-1-pyrrolidinyl]-carbonyl]oxy]-12-hydroxycholan-24-oicacid;(3α,5β,12α)-3-[[[-trans-3,4-bis[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-1-pyrrolidinyl]carbony]oxy]-12-hydroxycholan-24-oicacid;[3α[3(S),4(S)],5β12α]-3-[[[trans-3,4-bis[[[5-[[2-[bis(carboxy-methy)amino]-ethyl](carboxymetyl)amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbo-nyl]oxy]-12-hydroxycholan-24-oicacid;[3α[1(S),2(S)],5β,7α,12α]-3-[[[[cis-1,2-bis[[[5-[2-[bis(carboxmethyl)amino]amino]-5-carboxypentyl]amino]carbonyl]-4-cyclα-pentyl]amino]carbonyl]-oxy]-7,12-dihydroxycholan-24-oicacid;[3α[1(S)]5β,7α,12α]-3-[[[[cis-1-[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-5-carboxypentyl]amino]carbonyl]-2-[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacycylododec-1-yl]acetyl]amino]ethyl]amino]-carbonyl]-4-cyclo-pentyl]amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oicacid;[3β[3(S),5(S)],5β,7α,12α]-3-[[[3,5-bis[[4-[bis[2-[(bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxy-cholan-24-oicacid.
 7. A compound as claimed in claim 1 wherein the bi- or trivalentmetal ions complexed with the chelant residues Y₁ and Y₂ are selectedfrom Fe⁽²⁺⁾, Fe⁽³⁺⁾, Gd⁽³⁺⁾, Eu⁽³⁺⁾, Dy⁽³⁺⁾, La⁽³⁺⁾, Yb⁽³⁺⁾ and Mn⁽²⁺⁾.8. A compound as claimed in claim 1 wherein the salifying organic baseis selected from etbanolamine, diethanolamine, niorpholine, glucamine,N,N-dimethylglucamine, N-methylglucamine, lysine, arginine, ornithine.9. A compound as claimed in claim 1 wherein the anion of the salifyinginorganic acid is selected from halo acid ions such as iodides, bromidesand chlorides.
 10. A diagnostic contrastographic pharmaceuticalcomposition comprising at least one of the chelates as claimed in claim1 or a physiologically compatible salt thereof.
 11. method of magneticresonance imaging the organs or tissues of a human or animal bodycomprising providing a chelate of claim 1 or a physiologicallycompatible salt thereof and magnetic resonance imaging the subject tovisualize the organs or tissues.
 12. The method of claim 11 in which thevascular system is visualized.
 13. The method of claim 12 in which thecoronary vasculature is visualized.